Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: The roles of glutathione and glutathione peroxidase

Three different types of red blood cells (REC) were used:(i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH a nd (iii) REC from high-GSH sheep treated with CDNB to deplete GSH. Incubati on of these RBC with t-butyl hydroperoxide (tBHP, 3mM) for 10 min caused th e formation of TEARS, oxidation of haemoglobin and degradation and aggregat ion of membrane proteins in RBC from low-GSH sheep and GSH-depleted REC. By contrast, RBC from high-GSH sheep(normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreit ol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TEARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted REC or normal RBCs in the presence of 1. 5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degrada tion and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymati c system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known ant ioxidants, Butylated hydroxytoluene, ascorbate, cc-tocopherol and desferrio xamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insul t.